Folate (folic acid) is a vitamin that is essential for the life-sustaining processes of DNA synthesis, replication and repair. Folate is also important for protein biosynthesis, another process that is central to cell viability. The pteridine compound, methotrexate (MTX), is structurally similar to folate and as a result can bind to the active sites of a number of enzymes that normally use folate as a coenzyme for biosynthesis of purine and pyrimidine nucleotide precursors of DNA and for interconversion of amino acids during protein biosynthesis. Despite its structural similarity to folic acid, methotrexate cannot be used as a cofactor by enzymes that require folate, and instead competes with the folate cofactor for enzyme binding sites, thereby inhibiting protein and DNA biosynthesis and, hence, cell division.
The ability of methotrexate to inhibit cell division has been exploited in the treatment of a number of diseases and conditions that are characterized by rapid or aberrant cell growth. As an example, autoimmune diseases are characterized by an inappropriate immune response directed against normal autologous (self) tissues and are mediated by rapidly replicating T-cells or B-cells. Autoimmune diseases that have been treated with methotrexate include, without limitation, rheumatoid arthritis and other forms of arthritis, psoriasis, multiple sclerosis, the autoimmune stage of diabetes mellitus (juvenile-onset or Type 1 diabetes), autoimmune uveoretinitis, myasthenia gravis, autoimmune thyroiditis, and systemic lupus erythematosus.
Because many malignant cells proliferate more rapidly than normal cells, methotrexate can also be used to selectively impair cancerous cell growth. As a consequence, methotrexate is a widely used anticancer agent, employed, for example, in the treatment of acute lymphocytic leukemia, breast cancer, epidermoid cancers of the head and neck, advanced mycosis fungoides, lung cancer, non-Hodgkins lymphomas, gestational choriocarcinoma, chorioadenoma destruens, and hydatidiform moles.
Despite its therapeutic efficacy for a wide variety of diseases and conditions, treatment with methotrexate can present a risk to the patient. In particular, because methotrexate interferes with processes required for replication and division of normal as well as diseased cells, inappropriately high levels of the drug can lead to destruction of actively proliferating non-target tissues such as bone marrow and intestinal mucosa. Methotrexate has been associated with renal and hepatic toxicity when administered in the “high-dose regimen” that is required for some conditions. In addition, low-dose methotrexate therapy can lead to toxicity and unwanted side-effects in some patients, where the dosage is not appropriate due to individual variability in pharmacokinetic parameters influencing, for example, drug uptake, targeting and clearance. This situation is especially problematic in the treatment of chronic conditions such as rheumatoid arthritis, where methotrexate can be administered over a period of many years.
Because individual differences in pharmacokinetic parameters can be difficult to predict, safe and effective methotrexate treatment strategies require that methotrexate or methotrexate metabolite levels be monitored in patients being treated. A variety of methods have been developed for monitoring methotrexate drug concentrations in plasma including bioassays, immunological detection and chromatographic assays. Such plasma detection methods have been useful for monitoring high dose methotrexate therapy in some clinical applications. However, due to limitations in their sensitivity, these plasma detection methods have not been useful in monitoring low-dose methotrexate therapy, for which intracellular levels of methotrexate metabolites must be assayed.
Methotrexate is metabolized upon uptake by mammalian cells, such that one or more glutamyl moieties are added to methotrexate to yield a mixture of methotrexate polyglutamates (MTXPGs). The number of glutamyl moieties that can be added to MTX generally varies from two to seven. MTXPGs do not readily efflux from cells and thus are able to exert their cytotoxic effects over long periods of time. Levels of intracellular MTXPGs have been shown to be higher in patients that responded to methotrexate therapy as compared to intracellular levels in patients that did not respond. Currently available methods for measuring intracellular methotrexate polyglutamate levels are based on a dihydrofolate reductase enzyme assay in which methotrexate polyglutamate levels are calculated based on their ability to inhibit the dihydrofolate reductase enzyme. However, the extent of enzyme inhibition in these assays is dependent upon the number of glutamyl residues attached to methotrexate, rendering an accurate determination of intracellular methotrexate polyglutamate levels impossible by this method. The variability of dihydrofolate reductase based assays can be further exacerbated in some situations because folates, which are present in different amounts depending upon an individual's response to methotrexate therapy and the amount of folate contributed by diet, also influence the results of the current enzyme assay.
Thus, there exists a need for new methods for determining intracellular levels of methotrexate polyglutamates and for monitoring the efficacy and toxicity of methotrexate therapy including low-dose methotrexate therapy. The present invention satisfies this need and provides related advantages as well.